Cloning And Retroviral Transduction Of Mutant And Wt Her2 Into Cell Lines
HER2 somatic mutations were introduced into the pcDNA3 vector bearing the HER2 cDNA using the QuikChange II kit . These constructs were then shuttled into the pCFG5 retroviral vector, which contains a zeocin resistance marker and an IRES-GFP sequence, using the In-Fusion HD cloning system kit . The desired constructs were verified using Sanger sequencing. Retroviral particles were produced using ÏNX amphotrophic packaging cell line and cell lines were infected as per prior publications . Cell lines were subjected to 1 to 2 weeks of zeocin selection and transgene expression was verified by flow cytometry analysis for GFP expression and Western blot analysis for HER2. HER2 cell surface expression was measured by flow cytometry using a 1:20 dilution of anti-human HER2-APC . Data were collected at medium sheath pressure on a BD FACSCalibur custom modified by Cytek to contain a second excitation laser .
How Does Her2 Positive Breast Cancer Develop
While we are still learning about the causes of HER2 positive breast cancer, researchers have identified how HER2 positive breast cancer develops. In about 25 percent of breast cancers, the cancer cells have an excess of the HER2 protein. This is caused by a mutation in the HER2 gene. When the HER2 gene mutates, it causes cells in the breast to grow and divide at an uncontrolled rate, leading to tumor growth.
Somatic Mutations In Her2 And Implications For Current Treatment Paradigms In Her2
1Faculty of Biomedical Sciences and Health, Universidad Europea de Madrid, C/Tajo, S/N, 28670 Villaviciosa de Odón, Madrid, Spain
Breast cancer is the most common cancer type worldwide and is considered a heterogeneous genomic disease in terms of molecular markers, prognosis, and treatments . At the molecular level, at least five clinical subtypes have been defined: hormone receptor-positive , human epidermal growth factor receptor-2 , basal-like, normal-like, and triple-negative breast cancer . Based on this classification, the oncologist is able to prescribe the best endocrine therapy, chemotherapy , and/or HER2-targeted therapy. About 2025% of all breast cancers overexpress human epidermal growth factor receptor-2 and are referred to as HER2-positive. HER2 overexpression is linked to an aggressive phenotype resulting in reduced disease-free and overall survival compared with other breast cancer subtypes, and different strategies have been developed to try to block this receptor . According to clinical data, HER2-targeted therapy significantly improves the survival of breast cancer patients showing HER2 overexpression. However, recent data suggest the presence of oncogenic mutations in HER2 affects clinical outcome in HER2-positive breast cancer patients .
2.1. Mutations in HER2 Gene in Different Breast Cancer Histologies
2.2. Mutations in the Tyrosine Kinase Domain
in vitro.in vivo
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Why Do I Need Her2 Breast Cancer Testing
If you’ve been diagnosed with breast cancer, you may need this test to find out if your cancer is HER2-positive or HER2-negative. If you are already being treated for HER2-positive breast cancer, you may need this test to:
- Find out if your treatment is working. Normal levels of HER2 may mean you are responding to treatment. High levels may mean the treatment is not working.
- Find out if cancer has come back after treatment.
Effect On Tyrosine Kinase Activity And Cellular Signaling
In vitro kinase assays were conducted on the HER2 kinase domain mutations using recombinant expression of the isolated HER2 kinase domain . In this system, the activity of monomeric or dimeric HER2 can be compared . Wild-type HER2 showed a 2.3-fold increase in kinase activity upon in vitro dimerization . All 3 mutations tested here, HER2 V777L, D769H, and V842I, had greater tyrosine kinaseâspecific activity than wild-type , with further increases seen with in vitro dimer formation. While the V842I had the lowest activity of these 3 mutations, it did show a statistically significant increase over WT HER2. The L755S and del.755â759 mutations were poorly expressed and could not be assayed in this system.
The HER2 L755S was previously shown to produce lapatinib resistance in mammalian cells , and we, therefore, tested the lapatinib sensitivity of MCF10A-HER2 V777L and L755S cells. MCF10A-HER2 V777L cells were sensitive to lapatinib with reduced HER2 autophosphorylation seen at 50 nmol/L and complete inhibition observed with 500 nmol/L lapatinib . In contrast, L755S required doses of 1Î¼m lapatinib or more to inhibit HER2 signaling, but it was sensitive to the irreversible tyrosine kinase inhibitors, neratinib and canertinib . Complete inhibition of signaling by HER2 L755S was observed even at the lowest dose of neratinib used here .
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How Perjeta Is Thought To Work
Perjeta is an antibody that binds to HER2 and prevents the pairing of HER2 with other HER receptors. This helps block signaling that can lead to cancer growth.
It also recruits immune cells to help fight the cancer.
Because HER2 is present in all cells, Perjeta may also affect healthy cells.
Combining Perjeta and Herceptin is thought to provide a more comprehensive blockade of HER2 signaling.
According to Max Hasmann, a Roche scientist who was working on the project in Penzberg, Germany: It wasnt easy because in the beginning everyone would ask: Why do you need two antibodies targeting the same target? After all, they had already developed Herceptin for HER2-positive breast cancer. Did they really need another medicine for the same thing?
It was scientifically plausible. Not standard, but still plausible, says Sliwkowski. The approach was in stark contrast to how second-generation medicines are normally designed with the objective of the second outperforming the first. But if the success of Herceptin had proved anything, it was that they didnt always have to follow the standard way of doing things, they just had to follow their science.
Like Herceptin, Perjeta is associated with serious side effects and may cause heart problems. It carries a boxed warning because of these serious cardiac risks, and doctors must monitor patients heart function closely. Perjeta also has a boxed warning for the potential to harm an unborn baby.
What Is Her2 And What Does It Mean
HER2 is a protein that helps breast cancer cells grow quickly. Breast cancer cells with higher than normal levels of HER2 are called HER2-positive. These cancers tend to grow and spread faster than breast cancers that are HER2-negative, but are much more likely to respond to treatment with drugs that target the HER2 protein.
All invasive breast cancers should be tested for HER2 either on the biopsy sample or when the tumor is removed with surgery.
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Changes In Her2 Status After Neoadjuvant Chemotherapy
Neoadjuvant chemotherapy is currently considered as standard treatment for locally advanced breast cancer . Alteration of biomarker status after NAC is occasionally found in breast cancer . Hormone receptor status changed more often than HER2 status, and as for hormone receptors, positive to negative conversion was more common than negative to positive conversion . The frequency of HER2 change after NAC is reported in up to 15%, and both positive to negative conversion and negative to positive conversion were found with no preponderance . Previous studies on HER2 change after NAC are summarized in . In our study, HER2 status was altered after NAC in 3.4% with positive to negative conversion in 0.9% and negative to positive conversion in 2.5% . Most cases with negative to positive conversion of HER2 status after NAC showed low level of HER2 amplification, and the HER2/CEP17 ratio ranged from 2.2 to 4.4 . Cockburn et al. also reported the mean HER2/CEP17 ratio in resection specimens with HER2 positive conversion was 3.7. Although there are no guidelines about whether treatment should be modified based on altered biomarker status after NAC, the change of HER2 status may have an impact on the therapeutic management in certain patients. Accordingly, re-evaluation of biomarkers including HER2 after NAC is recommended for proper management.
Who Should Be Tested For Hboc
Most breast and ovarian cancers occur by chance with no known cause, so testing for BRCA1 or BRCA2 gene mutations may not be beneficial for the average person. Genetic testing is recommended primarily for people who have a personal and/or family history that suggests HBOC. However, women younger than 60 with triple-negative breast cancer , are at risk of having a BRCA mutation, regardless of family history.
For women with a previous diagnosis of breast cancer or ovarian cancer and/or a family history of breast or ovarian cancer, the National Comprehensive Cancer Network provides recommendations for when genetic counseling and testing may be needed. These recommendations are based on your familys history of cancer and how closely related you are to the person who developed cancer. Here are some important definitions to know:
“First-degree relatives” include parents, siblings, and children.
“Second-degree relatives” include aunts/uncles, grandparents, grandchildren, and nieces/nephews.
“Third-degree relatives” include first cousins, great-grandparents, or great-grandchildren.
Genetic testing should be considered if a person or family meets 1 or more of the criteria listed below:
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What Do The Test Results Mean
The results of HER2 testing will guide you and your cancer care team in making the best treatment decisions.
It is not clear if one test is more accurate than the other, but FISH is more expensive and takes longer to get the results. Often the IHC test is done first.
- If the IHC result is 0 or 1+, the cancer is considered HER2-negative. These cancers do not respond to treatment with drugs that target HER2.
- If the IHC result is 3+, the cancer is HER2-positive. These cancers are usually treated with drugs that target HER2.
- If the IHC result is 2+, the HER2 status of the tumor is not clear and is called “equivocal.” This means that the HER2 status needs to be tested with FISH to clarify the result.
Triple-negative breast tumors dont have too much HER2 and also dont have estrogen or progesterone receptors. They are HER2-, ER-, and PR-negative. Hormone therapy and drugs that target HER2 are not helpful in treating these cancers. See Triple-negative Breast Cancer to learn more.
Triple-positive breast tumorsare HER2-, ER-, and PR-positive. These cancers are treated with hormone drugs as well as drugs that target HER2.
Our team is made up of doctors and oncology certified nurses with deep knowledge of cancer care as well as journalists, editors, and translators with extensive experience in medical writing.
Last Revised: November 8, 2021
Inhibition Of Cell Growth By Neratinib And Lapatinib
Given the effectiveness of neratinib and lapatinib seen above, we measured the IC50 values of these 2 HER2/EGFR tyrosine kinase inhibitors on MCF10A-HER2 cells . BT474 cells, a HER2 geneâamplified cell line, served as the positive control and the IC50 value of less than 2 nmol/L with neratinib and 31 nmol/L with lapatinib match previously published values . MCF7 cells served as the negative control and were not sensitive to either inhibitor. MCF10A cells bearing mutant or WT HER2 were potently growth inhibited by neratinib . In contrast, lapatinib doses of 400 to 1,000 nmol/L were required to inhibit growth of most of these mutations. The MCF10A-HER2 L755S cells were resistant to lapatinib but could be readily inhibited by neratinib . Neratinib is an irreversible inhibitor of EGFR and HER2 and it has been used to treat gefitinib-resistant lung cancers that contain the EGFR T790M mutation .
Inhibition of cell growth by neratinib and lapatinib
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How Common Is Hboc
Most breast and ovarian cancers are sporadic, meaning they occur by chance with no known cause. Most women who have breast or ovarian cancer do not have HBOC.
Currently, it is estimated that less than 1% of the general population has a mutation in the BRCA1 or BRCA2 genes, and up to 10% of women and 20% of men diagnosed with breast cancer have a mutation in 1 of these genes. About 10% to 30% of women younger than 60 diagnosed with triple-negative breast cancer, which are cancers that do not have receptors for estrogen, progesterone, and HER2, have a BRCA1 or BRCA2 gene mutation, and others will have mutations in other breast cancer risk genes. Therefore, doctors recommend that women with triple-negative breast cancer receive genetic counseling and genetic testing .
HBOC is most common in families who have had multiple cases of breast cancer and/or ovarian cancer on the same side of the family. In families with 4 or more cases of breast cancer diagnosed before age 60, the chance of HBOC is about 80%. To compare, the chance of finding HBOC when only 1 woman has had breast cancer diagnosed before age 50 is estimated to be 10% or less.
Families with Ashkenazi Jewish ancestry have an increased chance of having HBOC. There are 3 specific gene mutations, known as founder mutations, that are common in these families:
185delAG in BRCA1
5382insC in BRCA1
6174delT in BRCA2
Methods Of Her2 Testing
Currently, immunohistochemistry , fluorescence in situ hybridization , and chromogenic in situ hybridization including silver in situ hybridization are regarded as standard methods for determination of HER2 status in breast cancer, and some of them have been approved by the U.S. Food and Drug Administration for HER2 testing in breast cancer since 1998.
Although HER2 status can be directly tested by in situ hybridization , many laboratories have adopted IHC as a screening test, and FISH as a confirmation test for HER2 IHC equivocal cases, considering higher failure rate, longer procedure time and higher reagent cost of FISH, compared to that of IHC. Moreover, high concordance has been found between HER2 protein overexpression by IHC and gene amplification by FISH . Finally, the 2007 ASCO/CAP guidelines stated that HER2 status should be initially assessed by IHC using a semi-quantitative scoring system , and confirmed by FISH in all IHC score 2+ equivocal cases .
Representative examples of human epidermal growth factor receptor 2 immunohistochemistry in breast cancer. HER2 IHC negative . HER2 IHC negative . HER2 IHC equivocal . HER2 IHC positive .
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How Is Hboc Inherited
Normally, each person has 2 copies of each gene in their bodys cells: 1 copy is inherited from a persons mother and 1 copy is inherited from a persons father. HBOC follows an autosomal dominant inheritance pattern. This means that a mutation needs to happen in only 1 copy of the gene for the person to have an increased risk of getting that disease. This means that a parent with a gene mutation may pass along a copy of their normal gene or a copy of the gene with the mutation. Therefore, a child who has a parent with a mutation has a 50% chance of inheriting that mutation. A sibling or parent of a person who has a mutation also has a 50% chance of having inherited the same mutation. However, if the parents test negative for the mutation , the risk to the siblings significantly decreases but their risk may still be higher than an average risk.
What Tests Can Detect Breast Cancer Her2 Status
Doctors will typically look for a HER2 defect with a biopsy, which is a procedure to remove and test a sample of tumor tissue.
The two main types of tests used to detect breast cancer HER2 status are:
- Immunohistochemistry assay . This test uses a chemical dye to stain the HER2 proteins. It measures the amount of these proteins on the surface of the cells.
- Fluorescence in situ hybridization test . With a FISH test, special labels with chemicals added to them change color and glow in the dark when they attach to HER2 proteins. It looks for extra copies of the HER2 gene.
The FISH test is the most accurate. But itâs also the most expensive, and it takes a while to get your results. For these reasons, your doctor might first suggest that you have an IHC test.
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How Are Breast Tumors Tested For Her2
Either a test called an immunohistochemistry test or fluorescence in situ hybridization test is used to find out if cancer cells have a high level of the HER2 protein.
See Testing Biopsy and Cytology Specimens for Cancer and Understanding Your Pathology Report: Breast Cancerto get more details about these tests.
Survival Rates And Statistics
A relative survival rate helps give an idea of how long a person with a particular condition will live after receiving a diagnosis compared with those without the condition.
For example, if the 5-year relative survival rate is 70%, it means that a person with the condition is 70% as likely to live for 5 years as someone without the condition.
It is important to remember that these figures are estimates. A person can talk with a doctor about how their condition is likely to affect them.
Some factors affecting a personâs survival rate with breast cancer include:
- individual factors, such as the personâs age and overall health
- the stage of the cancer at diagnosis
- the treatment the person receives
HER2-positive cancers are than HER2-negative cancers. With treatment, however, the chances of survival are high, especially with an early diagnosis. In some cases, they may be higher than for HER2-negative breast cancer due to effective targeted treatment.
According to the , the likelihood of living for another 5 years with HER2-positive cancer, compared with a person who does not have breast cancer, is as follows. These statistics are based on figures for the years 2011â2017.
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What Do The Results Mean
If HER2 protein levels are higher than normal or extra copies of the HER2 gene are found, it probably means you have HER2-positive cancer. If your results show normal amounts of HER2 protein or the normal number HER2 genes, you probably have HER2-negative cancer.
If your results were not clearly positive or negative, you will probably get retested, either using a different tumor sample or using a different testing method. Most often, IHC is done first, followed by FISH . IHC testing is less expensive and provides faster results than FISH. But most breast specialists think FISH testing is more accurate.
Treatments for HER2-positive breast cancer can substantially shrink cancerous tumors, with very few side effects. These treatments are not effective in HER2-negative cancers.
If you are being treated for HER2-positive cancer, normal results may mean you are responding to treatment. Results that show higher than normal amounts may mean your treatment is not working, or that cancer has come back after treatment.